POS0145 CLINICAL AND PATHOGENIC SIGNIFICANCE OF S100A4 OVEREXPRESSION IN SYSTEMIC SCLEROSIS
Authors: V. Ong, X. Shiwen, F. Zhang, R. Maclean, K. Clark, S. Borchert, R. I. Hussain, J. Klingelhöfer, J. Hallén, C. P. Denton
Journal: Annals of the Rheumatic Diseases (2023)

<h3>Background</h3> S100A4 belongs to a family of proteins originally defined by solubility of the prototypic members in 100% saturated ammonium sulphate. Preclinical studies suggest that extracellular S100A4 as a Damage Associated Molecular Pattern (DAMP) protein is upregulated upon stress or injury and may be a target for antifibrotic therapy. <h3>Objectives</h3> We explored expression and function of S100A4 as a driver of fibroblast activation in systemic sclerosis (SSc). <h3>Methods</h3> S100A4 protein concentration was measured by ELISA in serum of SSc (n=94) and healthy controls (n=15). Association of serum level with major organ complications and skin score (mRSS) was determined. S100A4 protein expression in supernatant and cell layer of skin fibroblast cultures from diffuse cutaneous SSc (SScF, n=6) and healthy controls (NF, n=6) was assessed. Recombinant S100A4 and a high affinity anti-S100A4 neutralising monoclonal antibody (AX-202) were used to investigate the profibrotic molecular, functional and genomic phenotype in SSc and control fibroblasts. <h3>Results</h3> Median [range] S100A4 (ng/ml) was higher in serum of SSc (89.9 [15.0-240.0]) than healthy controls (71.4 [7.9-131.8]; p=0.027). There was significant association with SSc-ILD (p=0.025, n=55), scleroderma renal crisis (p=0.026, n=4) and trend of increase in pulmonary hypertension (p=0.075, n=7) and a trend for S100A4 to fall in longer disease duration (r=-0.18, p=0.09) and a weak correlation (r=0.28, p=0.07) with current mRSS but not peak mRSS in dcSSc. Median [range] S100A4 (ng/ml) was higher in culture supernatants of SScF (4.19 [0.52-8.42]) than NF (0.28 [0.02-3.29]; p&lt;0.0001) and SSc cell layer had increased levels by Western blot analysis (mean [sd] RDU NF 0.50 [0.91]; SScF 8.16 [2.49]; p&lt;0.0001). S100A4 induced a fibrotic phenotype in NF with concentration-dependent increase in Col1, SMA and CTGF expression, and promotion of migration (scratch wound assay) and 3D type I collagen gel contraction that was attenuated by AX-202. AX-202 also reduced the constitutive profibrotic gene and protein expression phenotype of SScF. The effect of S100A4 was significantly attenuated by SB431524, a specific inhibitor of ALK5 (p&lt;0.0001 ANOVA) using qPCR assays across treatment groups for Col1, SMA and CTGF. Gene expression data (qPCR) confirmed protein expression. Genome-wide RNAseq analysis identified an S100A4 activated signature in NF overlapping the hallmark gene expression signature of SScF. Thus, 464 differentially expressed genes (FDR&lt;0.001 and FC&gt;1.5) induced in NF by S100A4, were also constitutively overexpressed, and downregulated by AX-202, in SScF (<b>Figure 1A</b>). Pathway mapping of these S100A4 dependent genes in SSc showed the most significant enriched Kegg pathways (FDR&lt;0.001) were regulation of stem cell pluripotency (4.6-fold) and metabolic pathways (1.9-fold) (<b>Figure 1B</b>). <h3>Conclusion</h3> Our findings provide compelling evidence supporting a profibrotic role for S100A4 in fibroblast activation in SSc and suggest that serum level may be a biomarker of major organ manifestations and disease severity. This study supports examining the therapeutic potential of targeting S100A4 in SSc are warranted. <h3>REFERENCES:</h3> NIL. <h3>Acknowledgements:</h3> NIL. <h3>Disclosure of Interests</h3> Voon Ong Speakers bureau: Boehringer Ingelheim, Xu Shiwen: None declared, Fenge Zhang: None declared, Rory Maclean: None declared, Kristina Clark: None declared, Signe Borchert Employee of: Arxx Therapeutics, Rizwan I Hussain Employee of: Arxx Therapeutics, Jörg Klingelhöfer Employee of: Arxx Therapeutics, Jonas Hallén Employee of: Arxx Therapeutics, Christopher P Denton Speakers bureau: Janssen; Boehringer Ingelheim, Consultant of: GSK; CSL Behring; Boehringer Ingelheim; Merck; Roche; Sanofi, Grant/research support from: GSK; CSL Behring; Inventiva; Horizon; Arxx Therapeutics.